Toll-like receptor 7 (TLR7) agonists having a pyridine or pyrazine moiety, conjugates thereof, and methods and uses therefor

ABSTRACT

Compounds having a structure according to formula (I) 
                         
where R 1  and Ar are as defined herein, are agonists for the Toll-like receptor 7 (TLR7) and can be used as adjuvants for stimulating the immune system. Some such compounds can be used in conjugates for targeted delivery to the organ or tissue of intended action.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application Ser. No. 62/546,195, filed Aug. 16, 2017; thedisclosure of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

This disclosure relates to Toll-like receptor 7 (“TLR7”) agonists andconjugates thereof, and methods for the preparation and use of suchagonists and their conjugates.

Toll-like receptors (“TLRs”) are cell-surface receptors that recognizepathogen-associated molecular patterns (“PAMPs”). Activation of a TLR bythe binding of a corresponding PAMP signals potential infection by apathogen and stimulates the immune system to fight the infection. Humanshave 11 TLRs, named TLR1 through TLR11.

The activation of a TLR—with TLR7 being the most studied—by an agonistcan have an adjuvant effect on the action of vaccines and immunotherapyagents in treating a variety of conditions other than actual pathogeninfection, by stimulating the immune response.

TLR7 recognizes PAMPs associated with single-stranded RNA viruses. Itsactivation induces secretion of Type I interferons such as IFNα and IFNβ(Lund et al. 2004). It has two binding sites, one for single strandedRNA ligands such as ssRNA40 (Berghöfer et al. 2007) and one forguanosine (Zhang et al. 2016).

TLR7 can bind to, and be activated by, guanosine-like synthetic agonistssuch as imiquimod, resiquimod, and gardiquimod, which are based on a1H-imidazo[4,5-c]quinoline scaffold.

Synthetic TLR7 agonists based on a pteridinone molecular scaffold arealso known, as exemplified by vesatolimod (Desai et al. 2015), which hasbeen in Phase 2 clinical trials. The potency of vesatolimod is reportedto be 100× less than that of the corresponding purine-8-one compound, asmeasured by IFN-α induction (Roethle et al. 2013).

Other synthetic TLR7 agonists are based on a purine-like scaffold,frequently according to formula (A):

where R, R′, and R″ are structural variables, with R″ typicallycontaining an unsubstituted or substituted aromatic or heteroaromaticring.

Disclosures of bioactive molecules having a purine-like and their usesin treating conditions such as fibrosis, inflammatory disorders, cancer,or pathogenic infections include: Akinbobuyi et al. 2015b and 2016;Barberis et al. 2012; Carson et al. 2014; Ding et al. 2016, 2017a, and2017b; Graupe et al. 2015; Hashimoto et al. 2009; Holldack et al. 2012;Isobe et al. 2009a and 2012; Jin et al. 2017a and 2017b; Peterson 2014;Pryde 2010; and Seifert 2015.

The group R″ can be pyridyl: Bonfanti et al. 2015a and 2015b; Halcomb etal. 2015; Hirota et al. 2000; Isobe et al. 2000, 2002, 2004, 2006,2009a, 2011, and 2012; Kasibhatla et al. 2007; Koga-Yamakawa et al.2013; Musmuca et al. 2009; Nakamura 2012; Ogita et al. 2007; and Yu etal. 2013.

Bonfanti et al. 2015b discloses TLR7 modulators in which the two ringsof a purine moiety are spanned by a macrocycle:

A TLR7 agonist can be conjugated to a partner molecule, which can be,for example, a phospholipid, a poly(ethylene glycol) (“PEG”), or anotherTLR (commonly TLR2). Exemplary disclosures include: Carson et al. 2013,2015, and 2016, Chan et al. 2009 and 2011, Lioux et al. 2016, Maj et al.2015, Ban et al. 2017; Vernejoul et al. 2014, and Zurawski et al. 2012.Conjugation to an antibody has also been disclosed: Akinbobuyi et al.2013 and 2015a, and Gadd et al. 2015. A frequent conjugation site is atthe R″ group of formula (A).

TLR7 agonists based on a 5H-pyrrolo[3,2-d]pyrimidine scaffold have alsobeen disclosed. See Cortez et al. 2017a and 2017b, McGowan et al. 2017,and Li et al. 2018.

Jensen et al. 2015 discloses the use of cationic lipid vehicles for thedelivery of TLR7 agonists.

Some TLR7 agonists, including resiquimod are dual TLR7/TLR8 agonists.See, for example, Beesu et al. 2017; Lioux et al. 2016; and Vernejoul etal. 2014.

TLR7 agonists based on a 5H-pyrrolo[3,2-d]pyrimidine scaffold have alsobeen disclosed. See Cortez et al. 2017a and 2017b, McGowan et al. 2017,and Li et al. 2018.

Full citations for the documents cited herein by first author orinventor and year are listed at the end of this specification.

BRIEF SUMMARY OF THE INVENTION

In one aspect, this specification provides compounds having a structureaccording to formula (I)

wherein

-   -   Ar is

-   -   R¹ is (C₁-C₅ alkyl)O, (C₁-C₂ alkyl)O(CH₂)₂₋₃O, (C₁-C₅        alkyl)C(═O)O, (C₁-C₅ alkyl)NH, (C₁-C₂ alkyl)O(CH₂)₂₋₃NH, or        (C₁-C₅ alkyl)C(═O)NH;    -   R² is, independently for each occurrence thereof, H, C₁-C₃        alkyl, halo, O(C₁-C₃ alkyl), CN, or NO₂; and    -   R³ and R⁴ are independently H; C₁-C₆ alkyl; (CH₂)₂₋₄OH;        (CH₂)₂₋₄O(C₁-C₃ alkyl); (CH₂)₂₋₄NH₂; (CH₂)₂₋₄NH(C₁-C₃ alkyl);        (CH₂)₂₋₄N(C₁-C₃ alkyl)₂; (CH₂)₁₋₃(aryl); (CH₂)₁₋₃(heteroaryl);        (CH₂)₂₋₄(OCH₂CH₂)₂₋₈(CH₂)₂₋₄NHBoc;

-   -    wherein a CH₂ group in the cycloaliphatic ring may be replaced        by O, S, NH, or N(C₁-C₃ alkyl) and the cycloaliphatic ring may        be substituted with C₁-C₃ alkyl, OH, O(C₁-C₃ alkyl), halo,        (CH₂)₀₋₃NH₂, or (CH₂)₀₋₃NH(C₁-C₃ alkyl);    -    or R³ and R⁴ combine with the nitrogen to which they are bonded        to form a cyclic amine of the structure

-   -    wherein a CH₂ group in the cycloaliphatic ring that is        separated from the amine nitrogen by at least two CH₂ groups may        be replaced by O, S, NH, or N(C₁-C₃ alkyl) and the cyclic amine        may be substituted with C₁-C₃ alkyl, OH, O(C₁-C₃ alkyl), halo,        (CH₂)₀₋₃NH₂, or (CH₂)₀₋₃NH(C₁-C₃ alkyl).

Compounds according to formula (I) have activity as TLR7 agonists andsome of them can be conjugated for targeted delivery to a target tissueor organ of intended action.

BRIEF DESCRIPTION OF THE DRAWING(S)

FIG. 1 shows a scheme for preparing compounds of this disclosure.

FIG. 2 and FIG. 3 show schemes for preparing yet other compounds of thisdisclosure.

FIG. 4 and FIG. 5 show schemes for the preparation of agonist-linkercompounds.

FIG. 6 is a representative graph showing the TLR7 agonism activity of acompound of this invention.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Antibody” means whole antibodies and any antigen binding fragment(i.e., “antigen-binding portion”) or single chain variants thereof. Awhole antibody is a protein comprising at least two heavy (H) chains andtwo light (L) chains inter-connected by disulfide bonds. Each heavychain comprises a heavy chain variable region (V_(H)) and a heavy chainconstant region comprising three domains, C_(H1), C_(H2) and C_(H3).Each light chain comprises a light chain variable region (V_(L) orV_(k)) and a light chain constant region comprising one single domain,C_(L). The V_(H) and V_(L) regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDRs), interspersed with more conserved framework regions (FRs). EachV_(H) and V_(L) comprises three CDRs and four FRs, arranged from amino-to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. The variable regions contain a binding domain thatinteracts with an antigen. The constant regions may mediate the bindingof the antibody to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system. An antibody is said to “specificallybind” to an antigen X if the antibody binds to antigen X with a K_(D) of5×10⁻⁸ M or less, more preferably 1×10⁻⁸ M or less, more preferably6×10⁻⁹ M or less, more preferably 3×10⁻⁹ M or less, even more preferably2×10⁻⁹ M or less. The antibody can be chimeric, humanized, or,preferably, human. The heavy chain constant region can be engineered toaffect glycosylation type or extent, to extend antibody half-life, toenhance or reduce interactions with effector cells or the complementsystem, or to modulate some other property. The engineering can beaccomplished by replacement, addition, or deletion of one or more aminoacids or by replacement of a domain with a domain from anotherimmunoglobulin type, or a combination of the foregoing.

“Antigen binding fragment” and “antigen binding portion” of an antibody(or simply “antibody portion” or “antibody fragment”) mean one or morefragments of an antibody that retain the ability to specifically bind toan antigen. It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody, suchas (i) a Fab fragment, a monovalent fragment consisting of the V_(L),V_(H), C_(L) and C_(H1) domains; (ii) a F(ab′)₂ fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fab′ fragment, which is essentially an Fabwith part of the hinge region (see, for example, Abbas et al., Cellularand Molecular Immunology, 6th Ed., Saunders Elsevier 2007); (iv) a Fdfragment consisting of the V_(H) and C_(H1) domains; (v) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (vi) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a V_(H) domain; (vii) an isolated complementaritydetermining region (CDR); and (viii) a nanobody, a heavy chain variableregion containing a single variable domain and two constant domains.Preferred antigen binding fragments are Fab, F(ab′)₂, Fab′, Fv, and Fdfragments. Furthermore, although the two domains of the Fv fragment,V_(L) and V_(H), are encoded by separate genes, they can be joined,using recombinant methods, by a synthetic linker that enables them to bemade as a single protein chain in which the V_(L) and V_(H) regions pairto form monovalent molecules (known as single chain Fv, or scFv); see,e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodiesare also encompassed within the term “antigen-binding portion” of anantibody.

Unless indicated otherwise—for example by reference to the linearnumbering in a SEQ ID NO: listing—references to the numbering of aminoacid positions in an antibody heavy or light chain variable region(V_(H) or V_(L)) are according to the Kabat system (Kabat et al.,“Sequences of proteins of immunological interest, 5th ed., Pub. No.91-3242, U.S. Dept. Health & Human Services, NIH, Bethesda, Md., 1991,hereinafter “Kabat”) and references to the numbering of amino acidpositions in an antibody heavy or light chain constant region (C_(H1),C_(H2), C_(H3), or C_(L)) are according to the EU index as set forth inKabat. See Lazar et al., US 2008/0248028 A1, the disclosure of which isincorporated herein by reference, for examples of such usage. Further,the ImMunoGeneTics Information System (IMGT) provides at its website atable entitled “IMGT Scientific Chart: Correspondence between CNumberings” showing the correspondence between its numbering system, EUnumbering, and Kabat numbering for the heavy chain constant region.

An “isolated antibody” means an antibody that is substantially free ofother antibodies having different antigenic specificities (e.g., anisolated antibody that specifically binds antigen X is substantiallyfree of antibodies that specifically bind antigens other than antigenX). An isolated antibody that specifically binds antigen X may, however,have cross-reactivity to other antigens, such as antigen X moleculesfrom other species. In certain embodiments, an isolated antibodyspecifically binds to human antigen X and does not cross-react withother (non-human) antigen X antigens. Moreover, an isolated antibody maybe substantially free of other cellular material and/or chemicals.

“Monoclonal antibody” or “monoclonal antibody composition” means apreparation of antibody molecules of single molecular composition, whichdisplays a single binding specificity and affinity for a particularepitope.

“Human antibody” means an antibody having variable regions in which boththe framework and CDR regions (and the constant region, if present) arederived from human germline immunoglobulin sequences. Human antibodiesmay include later modifications, including natural or syntheticmodifications. Human antibodies may include amino acid residues notencoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo). However, “human antibody” does not include antibodiesin which CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

“Human monoclonal antibody” means an antibody displaying a singlebinding specificity, which has variable regions in which both theframework and CDR regions are derived from human germline immunoglobulinsequences. In one embodiment, human monoclonal antibodies are producedby a hybridoma that includes a B cell obtained from a transgenicnonhuman animal, e.g., a transgenic mouse, having a genome comprising ahuman heavy chain transgene and a light chain transgene fused to animmortalized cell.

“Aliphatic” means a straight- or branched-chain, saturated orunsaturated, non-aromatic hydrocarbon moiety having the specified numberof carbon atoms (e.g., as in “C₃ aliphatic,” “C₁₋₅ aliphatic,” “C₁-C₅aliphatic,” or “C₁ to C₅ aliphatic,” the latter three phrases beingsynonymous for an aliphatic moiety having from 1 to 5 carbon atoms) or,where the number of carbon atoms is not explicitly specified, from 1 to4 carbon atoms (2 to 4 carbons in the instance of unsaturated aliphaticmoieties). A similar understanding is applied to the number of carbonsin other types, as in C₂₋₄ alkene, C₄-C₇ cycloaliphatic, etc. In asimilar vein, a term such as “(CH₂)₁₋₃” is to be understand as shorthandfor the subscript being 1, 2, or 3, so that such term represents CH₂,CH₂CH₂, and CH₂CH₂CH₂.

“Alkyl” means a saturated aliphatic moiety, with the same convention fordesignating the number of carbon atoms being applicable. By way ofillustration, C₁-C₄ alkyl moieties include, but are not limited to,methyl, ethyl, propyl, isopropyl, isobutyl, t-butyl, 1-butyl, 2-butyl,and the like. “Alkylene” means a divalent counterpart of an alkyl group,such as CH₂CH₂, CH₂CH₂CH₂, and CH₂CH₂CH₂CH₂.

“Alkenyl” means an aliphatic moiety having at least one carbon-carbondouble bond, with the same convention for designating the number ofcarbon atoms being applicable. By way of illustration, C₂-C₄ alkenylmoieties include, but are not limited to, ethenyl (vinyl), 2-propenyl(allyl or prop-2-enyl), cis-1-propenyl, trans-1-propenyl, E- (or Z-)2-butenyl, 3-butenyl, 1,3-butadienyl (but-1,3-dienyl) and the like.

“Alkynyl” means an aliphatic moiety having at least one carbon-carbontriple bond, with the same convention for designating the number ofcarbon atoms being applicable. By way of illustration, C₂-C₄ alkynylgroups include ethynyl (acetylenyl), propargyl (prop-2-ynyl),1-propynyl, but-2-ynyl, and the like.

“Cycloaliphatic” means a saturated or unsaturated, non-aromatichydrocarbon moiety having from 1 to 3 rings, each ring having from 3 to8 (preferably from 3 to 6) carbon atoms. “Cycloalkyl” means acycloaliphatic moiety in which each ring is saturated. “Cycloalkenyl”means a cycloaliphatic moiety in which at least one ring has at leastone carbon-carbon double bond. “Cycloalkynyl” means a cycloaliphaticmoiety in which at least one ring has at least one carbon-carbon triplebond. By way of illustration, cycloaliphatic moieties include, but arenot limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl,cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, and adamantyl.Preferred cycloaliphatic moieties are cycloalkyl ones, especiallycyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. “Cycloalkylene”means a divalent counterpart of a cycloalkyl group.

“Heterocycloaliphatic” means a cycloaliphatic moiety wherein, in atleast one ring thereof, up to three (preferably 1 to 2) carbons havebeen replaced with a heteroatom independently selected from N, O, or S,where the N and S optionally may be oxidized and the N optionally may bequaternized. Preferred cycloaliphatic moieties consist of one ring, 5-to 6-membered in size. Similarly, “heterocycloalkyl,”“heterocycloalkenyl,” and “heterocycloalkynyl” means a cycloalkyl,cycloalkenyl, or cycloalkynyl moiety, respectively, in which at leastone ring thereof has been so modified. Exemplary heterocycloaliphaticmoieties include aziridinyl, azetidinyl, 1,3-dioxanyl, oxetanyl,tetrahydrofuryl, pyrrolidinyl, piperidinyl, piperazinyl,tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydrothiopyranyl sulfone,morpholinyl, thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinylsulfone, 1,3-dioxolanyl, tetrahydro-1,1-dioxothienyl, 1,4-dioxanyl,thietanyl, and the like. “Heterocycloalkylene” means a divalentcounterpart of a heterocycloalkyl group.

“Alkoxy,” “aryloxy,” “alkylthio,” and “arylthio” mean —O(alkyl),—O(aryl), —S(alkyl), and —S(aryl), respectively. Examples are methoxy,phenoxy, methylthio, and phenylthio, respectively.

“Halogen” or “halo” means fluorine, chlorine, bromine or iodine, unlessa narrower meaning is indicated.+

“Aryl” means a hydrocarbon moiety having a mono-, bi-, or tricyclic ringsystem (preferably monocyclic) wherein each ring has from 3 to 7 carbonatoms and at least one ring is aromatic. The rings in the ring systemmay be fused to each other (as in naphthyl) or bonded to each other (asin biphenyl) and may be fused or bonded to non-aromatic rings (as inindanyl or cyclohexylphenyl). By way of further illustration, arylmoieties include, but are not limited to, phenyl, naphthyl,tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthracenyl, andacenaphthyl. “Arylene” means a divalent counterpart of an aryl group,for example 1,2-phenylene, 1,3-phenylene, or 1,4-phenylene.

“Heteroaryl” means a moiety having a mono-, bi-, or tricyclic ringsystem (preferably 5- to 7-membered monocyclic) wherein each ring hasfrom 3 to 7 carbon atoms and at least one ring is an aromatic ringcontaining from 1 to 4 heteroatoms independently selected from from N,O, or S, where the N and S optionally may be oxidized and the Noptionally may be quaternized. Such at least one heteroatom containingaromatic ring may be fused to other types of rings (as in benzofuranylor tetrahydroisoquinolyl) or directly bonded to other types of rings (asin phenylpyridyl or 2-cyclopentylpyridyl). By way of furtherillustration, heteroaryl moieties include pyrrolyl, furanyl, thiophenyl(thienyl), imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl,isothiazolyl, triazolyl, tetrazolyl, pyridyl, N-oxopyridyl, pyridazinyl,pyrimidinyl, pyrazinyl, quinolinyl, isoquinolynyl, quinazolinyl,cinnolinyl, quinozalinyl, naphthyridinyl, benzofuranyl, indolyl,benzothiophenyl, oxadiazolyl, thiadiazolyl, phenothiazolyl,benzimidazolyl, benzotriazolyl, dibenzofuranyl, carbazolyl,dibenzothiophenyl, acridinyl, and the like. “Heteroarylene” means adivalent counterpart of a heteroaryl group.

Where it is indicated that a moiety may be substituted, such as by useof “unsubstituted or substituted” or “optionally substituted” phrasingas in “unsubstituted or substituted C₁-C₅ alkyl” or “optionallysubstituted heteroaryl,” such moiety may have one or more independentlyselected substituents, preferably one to five in number, more preferablyone or two in number. Substituents and substitution patterns can beselected by one of ordinary skill in the art, having regard for themoiety to which the substituent is attached, to provide compounds thatare chemically stable and that can be synthesized by techniques known inthe art as well as the methods set forth herein. Where a moiety isidentified as being “unsubstituted or substituted” or “optionallysubstituted,” in a preferred embodiment such moiety is unsubstituted.

“Arylalkyl,” (heterocycloaliphatic)alkyl,” “arylalkenyl,” “arylalkynyl,”“biarylalkyl,” and the like mean an alkyl, alkenyl, or alkynyl moiety,as the case may be, substituted with an aryl, heterocycloaliphatic,biaryl, etc., moiety, as the case may be, with the open (unsatisfied)valence at the alkyl, alkenyl, or alkynyl moiety, for example as inbenzyl, phenethyl, N-imidazoylethyl, N-morpholinoethyl, and the like.Conversely, “alkylaryl,” “alkenylcycloalkyl,” and the like mean an aryl,cycloalkyl, etc., moiety, as the case may be, substituted with an alkyl,alkenyl, etc., moiety, as the case may be, for example as inmethylphenyl (tolyl) or allylcyclohexyl. “Hydroxyalkyl,” “haloalkyl,”“alkylaryl,” “cyanoaryl,” and the like mean an alkyl, aryl, etc.,moiety, as the case may be, substituted with one or more of theidentified substituent (hydroxyl, halo, etc., as the case may be).

For example, permissible substituents include, but are not limited to,alkyl (especially methyl or ethyl), alkenyl (especially allyl), alkynyl,aryl, heteroaryl, cycloaliphatic, heterocycloaliphatic, halo (especiallyfluoro), haloalkyl (especially trifluoromethyl), hydroxyl, hydroxyalkyl(especially hydroxyethyl), cyano, nitro, alkoxy, —O(hydroxyalkyl),—O(haloalkyl) (especially —OCF₃), —O(cycloalkyl), —O(heterocycloalkyl),—O(aryl), alkylthio, arylthio, ═O, ═NH, ═N(alkyl), ═NOH, ═NO(alkyl),—C(═O)(alkyl), —C(═O)H, —CO₂H, —C(═O)NHOH, —C(═O)O(alkyl),—C(═O)O(hydroxyalkyl), —C(═O)NH₂, —C(═O)NH(alkyl), —C(═O)N(alkyl)₂,—OC(═O)(alkyl), —OC(═O)(hydroxyalkyl), —OC(═O)O(alkyl),—OC(═O)O(hydroxyalkyl), —OC(═O)NH₂, —OC(═O)NH(alkyl), —OC(═O)N(alkyl)₂,azido, —NH₂, —NH(alkyl), —N(alkyl)₂, —NH(aryl), —NH(hydroxyalkyl),—NHC(═O)(alkyl), —NHC(═O)H, —NHC(═O)NH₂, —NHC(═O)NH(alkyl),—NHC(═O)N(alkyl)₂, —NHC(═NH)NH₂, —OSO₂(alkyl), —SH, —S(alkyl), —S(aryl),—S(cycloalkyl), —S(═O)alkyl, —SO₂(alkyl), —SO₂NH₂, —SO₂NH(alkyl),—SO₂N(alkyl)₂, and the like.

Where the moiety being substituted is an aliphatic moiety, preferredsubstituents are aryl, heteroaryl, cycloaliphatic, heterocycloaliphatic,halo, hydroxyl, cyano, nitro, alkoxy, —O(hydroxyalkyl), —O(haloalkyl),—O(cycloalkyl), —O(heterocycloalkyl), —O(aryl), alkylthio, arylthio, ═O,═NH, ═N(alkyl), ═NOH, ═NO(alkyl), —CO₂H, —C(═O)NHOH, —C(═O)O(alkyl),—C(═O)O(hydroxyalkyl), —C(═O)NH₂, —C(═O)NH(alkyl), —C(═O)N(alkyl)₂,—OC(═O)(alkyl), —OC(═O)(hydroxyalkyl), —OC(═O)O(alkyl),—OC(═O)O(hydroxyalkyl), —OC(═O)NH₂, —OC(═O)NH(alkyl), —OC(═O)N(alkyl)₂,azido, —NH₂, —NH(alkyl), —N(alkyl)₂, —NH(aryl), —NH(hydroxyalkyl),—NHC(═O)(alkyl), —NHC(═O)H, —NHC(═O)NH₂, —NHC(═O)NH(alkyl),—NHC(═O)N(alkyl)₂, —NHC(═NH)NH₂, —OSO₂(alkyl), —SH, —S(alkyl), —S(aryl),—S(═O)alkyl, —S(cycloalkyl), —SO₂(alkyl), —SO₂NH₂, —SO₂NH(alkyl), and—SO₂N(alkyl)₂. More preferred substituents are halo, hydroxyl, cyano,nitro, alkoxy, —O(aryl), ═O, ═NOH, ═NO(alkyl), —OC(═O)(alkyl),—OC(═O)O(alkyl), —OC(═O)NH₂, —OC(═O)NH(alkyl), —OC(═O)N(alkyl)₂, azido,—NH₂, —NH(alkyl), —N(alkyl)₂, —NH(aryl), —NHC(═O)(alkyl), —NHC(═O)H,—NHC(═O)NH₂, —NHC(═O)NH(alkyl), —NHC(═O)N(alkyl)₂, and —NHC(═NH)NH₂.Especially preferred are phenyl, cyano, halo, hydroxyl, nitro,C₁-C₄alkyoxy, O(C₂-C₄ alkylene)OH, and O(C₂-C₄ alkylene)halo.

Where the moiety being substituted is a cycloaliphatic,heterocycloaliphatic, aryl, or heteroaryl moiety, preferred substituentsare alkyl, alkenyl, alkynyl, halo, haloalkyl, hydroxyl, hydroxyalkyl,cyano, nitro, alkoxy, —O(hydroxyalkyl), —O(haloalkyl), —O(aryl),—O(cycloalkyl), —O(heterocycloalkyl), alkylthio, arylthio,—C(═O)(alkyl), —C(═O)H, —CO₂H, —C(═O)NHOH, —C(═O)O(alkyl),—C(═O)O(hydroxyalkyl), —C(═O)NH₂, —C(═O)NH(alkyl), —C(═O)N(alkyl)₂,—OC(═O)(alkyl), —OC(═O)(hydroxyalkyl), —OC(═O)O(alkyl),—OC(═O)O(hydroxyalkyl), —OC(═O)NH₂, —OC(═O)NH(alkyl), —OC(═O)N(alkyl)₂,azido, —NH₂, —NH(alkyl), —N(alkyl)₂, —NH(aryl), —NH(hydroxyalkyl),—NHC(═O)(alkyl), —NHC(═O)H, —NHC(═O)NH₂, —NHC(═O)NH(alkyl),—NHC(═O)N(alkyl)₂, —NHC(═NH)NH₂, —OSO₂(alkyl), —SH, —S(alkyl), —S(aryl),—S(cycloalkyl), —S(═O)alkyl, —SO₂(alkyl), —SO₂NH₂, —SO₂NH(alkyl), and—SO₂N(alkyl)₂. More preferred substituents are alkyl, alkenyl, halo,haloalkyl, hydroxyl, hydroxyalkyl, cyano, nitro, alkoxy,—O(hydroxyalkyl), —C(═O)(alkyl), —C(═O)H, —CO₂H, —C(═O)NHOH,—C(═O)O(alkyl), —C(═O)O(hydroxyalkyl), —C(═O)NH₂, —C(═O)NH(alkyl),—C(═O)N(alkyl)₂, —OC(═O)(alkyl), —OC(═O)(hydroxyalkyl), —OC(═O)O(alkyl),—OC(═O)O(hydroxyalkyl), —OC(═O)NH₂, —OC(═O)NH(alkyl), —OC(═O)N(alkyl)₂,—NH₂, —NH(alkyl), —N(alkyl)₂, —NH(aryl), —NHC(═O)(alkyl), —NHC(═O)H,—NHC(═O)NH₂, —NHC(═O)NH(alkyl), —NHC(═O)N(alkyl)₂, and —NHC(═NH)NH₂.Especially preferred are C₁-C₄ alkyl, cyano, nitro, halo, andC₁-C₄alkoxy.

Where a range is stated, as in “C₁-C₅ alkyl” or “5 to 10%,” such rangeincludes the end points of the range, as in C₁ and C₅ in the firstinstance and 5% and 10% in the second instance.

Unless particular stereoisomers are specifically indicated (e.g., by abolded or dashed bond at a relevant stereocenter in a structuralformula, by depiction of a double bond as having E or Z configuration ina structural formula, or by use stereochemistry-designatingnomenclature), all stereoisomers are included within the scope of theinvention, as pure compounds as well as mixtures thereof. Unlessotherwise indicated, individual enantiomers, diastereomers, geometricalisomers, and combinations and mixtures thereof are all encompassed bythis invention.

Those skilled in the art will appreciate that compounds may havetautomeric forms (e.g., keto and enol forms), resonance forms, andzwitterionic forms that are equivalent to those depicted in thestructural formulae used herein and that the structural formulaeencompass such tautomeric, resonance, or zwitterionic forms.

“Pharmaceutically acceptable ester” means an ester that hydrolyzes invivo (for example in the human body) to produce the parent compound or asalt thereof or has per se activity similar to that of the parentcompound. Suitable esters include C₁-C₅ alkyl, C₂-C₅ alkenyl or C₂-C₅alkynyl esters, especially methyl, ethyl or n-propyl.

“Pharmaceutically acceptable salt” means a salt of a compound suitablefor pharmaceutical formulation. Where a compound has one or more basicgroups, the salt can be an acid addition salt, such as a sulfate,hydrobromide, tartrate, mesylate, maleate, citrate, phosphate, acetate,pamoate (embonate), hydroiodide, nitrate, hydrochloride, lactate,methylsulfate, fumarate, benzoate, succinate, mesylate, lactobionate,suberate, tosylate, and the like. Where a compound has one or moreacidic groups, the salt can be a salt such as a calcium salt, potassiumsalt, magnesium salt, meglumine salt, ammonium salt, zinc salt,piperazine salt, tromethamine salt, lithium salt, choline salt,diethylamine salt, 4-phenylcyclohexylamine salt, benzathine salt, sodiumsalt, tetramethylammonium salt, and the like. Polymorphic crystallineforms and solvates are also encompassed within the scope of thisinvention.

In the formulae of this specification, a wavy line (

) transverse to a bond or an asterisk (*) at the end of the bond denotesa covalent attachment site. For instance, a statement that R is

or that R is

in the formula

means

In the formulae of this specification, a bond traversing an aromaticring between two carbons thereof means that the group attached to thebond may be located at any of the positions of the aromatic ring madeavailable by removal of the hydrogen that is implicitly there. By way ofillustration, the formula

represents

In another illustration,

represents

Generally, tautomeric structures have been rendered herein in the enolform, as a matter of consistency and convenience.

Those skilled in the art will appreciate that they could also have berendered in the equivalent keto form and that the two tautomersequivalent.TLR7 Agonists

R¹ in formula (I) preferably is n-BuO, n-BuNH, EtO, MeO, or MeOCH₂CH₂O;more preferably n-BuO or MeOCH₂CH₂O; and most preferably n-BuO.

In one embodiment, a compound according to formula I is represented byformula (Ia), where R¹ is n-BuO or MeOCH₂CH₂O, preferably n-BuO:

Examples of compounds according to formula (Ia) include:

Table A presents biological activity data for compounds (Ia) disclosedherein. One set of data relates TLR7 agonism activity using theHEK-Blue™ TLR7 reporter assay, as described hereinbelow. Another set ofdata relates to the induction of interleukin 6 (IL-6), a cytokine thatplays an important role in the TLR7 pathway. For comparison, theactivities of resiquimod, vesatolimod, gardiquimod, and Compound B (CASReg. No. 226906-84-9) are also presented.

TLR7 Agonism IL-6 Induction Compound (EC₅₀, nM) (EC₅₀, μM) Resiquimod~230-430 — Vesatolimod 1,200 — Gardiquimod 3,340 — Compound B 470 —Ia-01 330 1.0  Ia-02 11,200 — Ia-03 180 1.0  Ia-04 310 0.60 Ia-05 1400.36 Ia-06 83 0.21 Ia-07 490 1.0  Ia-08 24 0.22 Ia-09 89 0.42 Ia-10 2900.57 Ia-11 770 — Ia-12 300 —

In another embodiment, a compound according to formula I is representedby formula (Ib), where R¹ is n-BuO or MeOCH₂CH₂O, preferably n-BuO:

Examples of compounds according to formula (Ib) include:

Table B presents biological activity data for compounds (Ib) disclosedherein.

TABLE B Biological Activity of Compounds (Ib) TLR7 Agonism IL-6Induction Compound (EC₅₀, nM) (EC₅₀, μM) Ib-01 15 0.077 Ib-02 28 0.11Ib-03 18 0.062 Ib-04 12 0.078 Ib-05 62 0.26 Ib-06 22 0.19 Ib-07 41 0.10Ib-08 47 0.33

In another embodiment, a compound according to formula I is representedby formula (Ib), where R¹ is n-BuO or MeOCH₂CH₂O, preferably n-BuO:

Examples of compounds according to formula (Ic) include:

Table C presents biological activity data for compounds (Ic).

TABLE C Biological Activity of Compounds (Ic) TLR7 Agonism IL-6Induction Compound (EC₅₀, nM) (EC₅₀, μM) Ic-01 4.8 0.026 Ic-02 9 — Ic-035.3 0.014 Ic-04 3.6 0.022 Ic-05 14 0.028 Ic-06 110 0.18 Ic-07 11 0.020Ic-08 23 0.15 Ic-09 11 0.037 Ic-10 6.1 0.027 Ic-11 2.9 0.023 Ic-12 3.10.013 Ic-13 4.9 0.011

In formulae (I), (Ia), (Ib), and (Ic), preferably R⁴ is H and R³ isother than H.

Specific examples of —N(R³)(R⁴) that can be used in compounds of formula(I), (Ia). (Ib), and (Ic) include:

ConjugatesGeneral

TLR7 agonists disclosed herein can be delivered to the site of intendedaction by localized administration or by targeted delivery in aconjugate with a targeting moiety.

Preferably, the targeting moiety is an antibody or antigen bindingportion thereof and its antigen is found at the locality of intendedaction, for example a tumor associated antigen if the intended site ofaction is at a tumor (cancer). Preferably, the tumor associated antigenis uniquely expressed or overexpressed by the cancer cell, compared to anormal cell. The tumor associated antigen can be located on the surfaceof the cancer cell or secreted by the cancer cell into its environs.

In one aspect, there is provided a conjugate comprising compound of thisinvention and a ligand, represented by formula (II)[D(X^(D))_(a)(C)_(c)(X^(Z))_(b)]_(m)Z  (II)where Z is a targeting moiety, D is an agonist of this invention, and—(X^(D))_(a)C(X^(Z))_(b)— are collectively referred to as a “linkermoiety” or “linker” because they link Z and D. Within the linker, C is acleavable group designed to be cleaved at or near the site of intendedbiological action of D; X^(D) and X^(Z) are spacer moieties (or“spacers”) that space apart D and C and C and Z, respectively;subscripts a, b, and c are independently 0 or 1 (that is, the presenceof X^(D), X^(Z) and C are optional). Subscript m is 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 (preferably 1, 2, 3, or 4). D, X^(D), C, X^(Z) and Z aremore fully described hereinbelow.

By binding to a target tissue or cell where its antigen or receptor islocated, Z directs the conjugate there. Cleavage of group C at thetarget tissue or cell releases D to exert its effect locally. In thismanner, precise delivery of D is achieved at the site of intendedaction, reducing the dosage needed. Also, D is normally biologicallyinactive (or significantly less active) in its conjugated state, therebyreducing off-target effects.

As reflected by the subscript m, each Z can conjugate with more than oneD, depending on the number of sites Z has available for conjugation andthe experimental conditions employed. Those skilled in the art willappreciate that, while each individual Z is conjugated to an integernumber of Ds, a preparation of the conjugate may analyze for anon-integer ratio of D to Z, reflecting a statistical average. Thisratio is referred to as the substitution ratio (“SR”) or thedrug-antibody ratio (“DAR”).

Targeting Moiety Z

Preferably, targeting moiety Z is an antibody. For convenience andbrevity and not by way of limitation, the detailed discussion in thisspecification about Z and its conjugates is written in the context ofits being an antibody, but those skilled in the art will understand thatother types of Z can be conjugated, mutatis mutandis. For example,conjugates with folic acid as the targeting moiety can target cellshaving the folate receptor on their surfaces (Leamon et al., Cancer Res.2008, 68 (23), 9839). For the same reasons, the detailed discussion inthis specification is primarily written in terms of a 1:1 ratio of Z toD (m=1).

Antibodies that can be used in conjugates of this invention includethose recognizing the following antigens: mesothelin, prostate specificmembrane antigen (PSMA), CD19, CD22, CD30, CD70, B7H3, B7H4 (also knownas 08E), protein tyrosine kinase 7 (PTK7), glypican-3, RG1, fucosyl-GM1,CTLA-4, and CD44. The antibody can be animal (e.g., murine), chimeric,humanized, or, preferably, human. The antibody preferably is monoclonal,especially a monoclonal human antibody. The preparation of humanmonoclonal antibodies against some of the aforementioned antigens isdisclosed in Korman et al., U.S. Pat. No. 8,609,816 B2 (2013; B7H4, alsoknown as 08E; in particular antibodies 2A7, 1G11, and 2F9); Rao-Naik etal., U.S. Pat. No. 8,097,703 B2 (2012; CD19; in particular antibodies5G7, 13F1, 46E8, 21D4, 21D4a, 47G4, 27F3, and 3C10); King et al., U.S.Pat. No. 8,481,683 B2 (2013; CD22; in particular antibodies 12C5, 19A3,16F7, and 23C6); Keler et al., U.S. Pat. No. 7,387,776 B2 (2008; CD30;in particular antibodies 5F11, 2H9, and 17G1); Terrett et al., U.S. Pat.No. 8,124,738 B2 (2012; CD70; in particular antibodies 2H5, 10B4, 8B5,18E7, and 69A7); Korman et al., U.S. Pat. No. 6,984,720 B1 (2006;CTLA-4; in particular antibodies 10D1, 4B6, and 1E2); Korman et al.,U.S. Pat. No. 8,008,449 B2 (2011; PD-1; in particular antibodies 17D8,2D3, 4H1, 5C4, 4A11, 7D3, and 5F4); Huang et al., US 2009/0297438 A1(2009; PSMA. in particular antibodies 1C3, 2A10, 2F5, 2C6); Cardarelliet al., U.S. Pat. No. 7,875,278 B2 (2011; PSMA; in particular antibodies4A3, 7F12, 8C12, 8A11, 16F9, 2A10, 2C6, 2F5, and 1C3); Terrett et al.,U.S. Pat. No. 8,222,375 B2 (2012; PTK7; in particular antibodies 3G8,4D5, 12C6, 12C6a, and 7C8); Harkins et al., U.S. Pat. No. 7,335,748B2(2008; RG1; in particular antibodies A, B, C, and D); Terrett et al.,U.S. Pat. No. 8,268,970 B2 (2012; mesothelin; in particular antibodies3C10, 6A4, and 7B1); Xu et al., US 2010/0092484 A1 (2010; CD44; inparticular antibodies 14G9.B8.B4, 2D1.A3.D12, and 1A9.A6.B9); Deshpandeet al., U.S. Pat. No. 8,258,266 B2 (2012; IP10; in particular antibodies1D4, 1E1, 2G1, 3C4, 6A5, 6A8, 7C10, 8F6, 10A12, 10A12S, and 13C4); Kuhneet al., U.S. Pat. No. 8,450,464 B2 (2013; CXCR4; in particularantibodies F7, F9, D1, and E2); and Korman et al., U.S. Pat. No.7,943,743 B2 (2011; PD-L1; in particular antibodies 3G10, 12A4, 10A5,5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4); the disclosures of whichare incorporated herein by reference. Preferably, the antibody is ananti-mesothelin antibody.

In addition to being an antibody, Z can also be an antibody fragment(such as Fab, Fab′, F(ab′)₂, Fd, or Fv) or antibody mimetic, such as anaffibody, a domain antibody (dAb), a nanobody, a unibody, a DARPin, ananticalin, a versabody, a duocalin, a lipocalin, or an avimer.

Any one of several different reactive groups on Z can be a conjugationsite, including ε-amino groups in lysine residues, pendant carbohydratemoieties, carboxylic acid groups on aspartic or glutamic acid sidechains, cysteine-cysteine disulfide groups, and cysteine thiol groups.For reviews on antibody reactive groups suitable for conjugation, see,e.g., Garnett, Adv. Drug Delivery Rev. 2001, 53, 171-216 and Dubowchikand Walker, Pharmacology & Therapeutics 1999, 83, 67-123, thedisclosures of which are incorporated herein by reference.

Most antibodies have multiple lysine residues, which can be conjugatedvia their ε-amino groups via amide, urea, thiourea, or carbamate bonds.

A thiol (—SH) group in the side chain of a cysteine can be used to forma conjugate by several methods. It can be used to form a disulfide bondbetween it and a thiol group on the linker. Another method is via itsMichael addition to a maleimide group on the linker.

Typically, although antibodies have cysteine residues, they lack freethiol groups because all their cysteines are engaged in intra- orinter-chain disulfide bonds. To generate a free thiol group, a nativedisulfide group can be reduced. See, e.g., Packard et al., Biochemistry1986, 25, 3548; King et al., Cancer Res. 1994, 54, 6176; and Doronina etal., Nature Biotechnol. 2003, 21, 778. Alternatively, a cysteine havinga free —SH group can be introduced by mutating the antibody,substituting a cysteine for another amino acid or inserting one into thepolypeptide chain. See, for example, Eigenbrot et al., U.S. Pat. No.7,521,541 B2 (2009); Chilkoti et al., Bioconjugate Chem. 1994, 5, 504;Urnovitz et al., U.S. Pat. No. 4,698,420 (1987); Stimmel et al., J.Biol. Chem. 2000, 275, 30445; Bam et al., U.S. Pat. No. 7,311,902 B2(2007); Kuan et al., J. Biol. Chem. 1994, 269, 7610; Poon et al., J.Biol. Chem. 1995, 270, 8571; Junutula et al., Nature Biotechnology 2008,26, 925 and Rajpal et al., U.S. Provisional Application No. 62/270,245,filed Dec. 21, 2015. In yet another approach, a cysteine is added to theC-terminus of the heavy of light chain. See, e.g., Liu et al., U.S. Pat.No. 8,865,875 B2 (2014); Cumber et al., J. Immunol. 1992, 149, 120; Kinget al, Cancer Res. 1994, 54, 6176; Li et al., Bioconjugate Chem. 2002,13, 985; Yang et al., Protein Engineering 2003, 16, 761; and Olafson etal., Protein Engineering Design & Selection 2004, 17, 21. Thedisclosures of the documents cited in this paragraph are incorporatedherein by reference.

Linkers and their Components

As noted above, the linker comprises up to three elements: a cleavablegroup C and optional spacers X^(Z) and X^(D).

Group C is cleavable under physiological conditions. Preferably it isrelatively stable while the conjugate is in circulation in the blood,but is readily cleaved once the conjugate reaches its site of intendedaction.

A preferred group C is a peptide that is cleaved selectively by aprotease inside the target cell, as opposed to by a protease in theserum. Typically, the peptide comprises from 1 to 20 amino acids,preferably from 1 to 6 amino acids, more preferably from 2 to 3 aminoacids. The amino acid(s) can be natural and/or non-natural α-aminoacids. Natural amino acids are those encoded by the genetic code, aswell as amino acids derived therefrom, e.g., hydroxyproline,γ-carboxyglutamate, citrulline, and O-phosphoserine. In thisspecification, the term “amino acid” also includes amino acid analogsand mimetics. Analogs are compounds having the same general H₂N(R)CHCO₂Hstructure of a natural amino acid, except that the R group is not onefound among the natural amino acids. Examples of analogs includehomoserine, norleucine, methionine-sulfoxide, and methionine methylsulfonium. An amino acid mimetic is a compound that has a structuredifferent from the general chemical structure of an α-amino acid butfunctions in a manner similar to one. The amino acid can be of the “L”stereochemistry of the genetically encoded amino acids, as well as ofthe enantiomeric “D” stereochemistry.

Preferably, C contains an amino acid sequence that is a cleavagerecognition sequence for a protease. Many cleavage recognition sequencesare known in the art. See, e.g., Matayoshi et al. Science 247: 954(1990); Dunn et al. Meth. Enzymol. 241: 254 (1994); Seidah et al. Meth.Enzymol. 244: 175 (1994); Thornberry, Meth. Enzymol. 244: 615 (1994);Weber et al. Meth. Enzymol. 244: 595 (1994); Smith et al. Meth. Enzymol.244: 412 (1994); and Bouvier et al. Meth. Enzymol. 248: 614 (1995); thedisclosures of which are incorporated herein by reference.

A group C can be chosen such that it is cleaved by a protease present inthe extracellular matrix in the vicinity of a cancer, e.g., a proteasereleased by nearby dying cancer cells or a tumor-associated proteasesecreted by cancer cells. Exemplary extracellular tumor-associatedproteases are plasmin, matrix metalloproteases (MMP), thimetoligopeptidase (TOP) and CD10. See, e.g., Trouet et al., U.S. Pat. No.7,402,556 B2 (2008); Dubois et al., U.S. Pat. No. 7,425,541 B2 (2008);and Bebbington et al., U.S. Pat. No. 6,897,034 B2 (2005). Cathepsin D,normally lysosomal enzyme found inside cells, is sometimes found in theenvirons of a tumor, possibly released by dying cancer cells.

For conjugates designed to be by an enzyme, C preferably comprises anamino acid sequence selected for cleavage by proteases such cathepsinsB, C, D, H, L and S, especially cathepsin B. Exemplary cathepsin Bcleavable peptides include Val-Ala, Val-Cit, Val-Lys, Lys-Val-Ala,Asp-Val-Ala, Val-Ala, Lys-Val-Cit, Ala-Val-Cit, Val-Gly, Val-Gln, andAsp-Val-Cit. (Herein, amino acid sequences are written in the N-to-Cdirection, as in H2N-AA²-AA¹-CO₂H, unless the context clearly indicatesotherwise.) See Dubowchik et al., Biorg. Med. Chem. Lett. 1998, 8, 3341;Dubowchik et al., Bioorg. Med. Chem. Lett. 1998, 8, 3347; and Dubowchiket al., Bioconjugate Chem. 2002, 13, 855; the disclosures of which areincorporated by reference.

Another enzyme that can be utilized for cleaving peptidyl linkers islegumain, a lysosomal cysteine protease that preferentially cleaves atAla-Ala-Asn.

In one embodiment, Group C is a peptide comprising a two-amino acidsequence -AA²-AA′- wherein AA′ is lysine, arginine, or citrulline andAA² is phenylalanine, valine, alanine, leucine or isoleucine. In anotherembodiment, C consists of a sequence of one to three amino acids,selected from the group consisting of Val-Cit, Ala-Val, Val-Ala-Val,Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Cit-Cit, Val-Lys, Ala-Ala-Asn, Lys,Cit, Ser, and Glu. More preferably, it is a two to three amino acidpeptide from the foregoing group.

The preparation and design of cleavable groups C consisting of a singleamino acid is disclosed in Chen et al., U.S. Pat. No. 8,664,407 B2(2014), the disclosure of which is incorporated herein by reference.

Group C can be bonded directly to Z or D; i.e. spacers X^(Z) or X^(D),as the case may be, can be absent.

When present, spacer X^(Z) provides spatial separation between C and Z,lest the former sterically interfere with antigen binding by latter orthe latter sterically interfere with cleavage of the former. Further,spacer X^(Z) can be used to confer increased solubility or decreasedaggregation properties to conjugates. A spacer X^(Z) can comprise one ormore modular segments, which can be assembled in any number ofcombinations. Examples of suitable segments for a spacer X^(Z) are:

and combinations thereof,where the subscript g is 0 or 1 and the subscript h is 1 to 24,preferably 2 to 4. These segments can be combined, such as illustratedbelow:

Spacer X^(D), if present, provides spatial separation between C and D,lest the latter interfere sterically or electronically with cleavage ofthe former. Spacer X^(D) also can serve to introduce additionalmolecular mass and chemical functionality into a conjugate. Generally,the additional mass and functionality will affect the serum half-lifeand other properties of the conjugate. Thus, through judicious selectionof spacer groups, the serum half-live of a conjugate can be modulated.Spacer X^(D) also can be assembled from modular segments, analogously tothe description above for spacer X^(Z).

Spacers X^(Z) and/or X^(D), where present, preferably provide a linearseparation of from 4 to 25 atoms, more preferably from 4 to 20 atoms,between Z and C or D and C, respectively.

The linker can perform other functions in addition to covalently linkingthe antibody and the drug. For instance, the linker can contain apoly(ethylene glycol) (“PEG”) group. Since the conjugation steptypically involves coupling a drug-linker to an antibody in an aqueousmedium, a PEG group many enhance the aqueous solubility of thedrug-linker. Also, a PEG group may enhance the solubility or reduceaggregation in the resulting ADC. Where a PEG group is present, it maybe incorporated into either spacer X^(Z) of X^(D), or both. The numberof repeat units in a PEG group can be from 2 to 20, preferably between 4and 10.

Either spacer X^(Z) or X^(D), or both, can comprise a self-immolatingmoiety. A self-immolating moiety is a moiety that (1) is bonded to C andeither Z or D and (2) has a structure such that cleavage from group Cinitiates a reaction sequence resulting in the self-immolating moietydisbonding itself from Z or D, as the case may be. In other words,reaction at a site distal from Z or D (cleavage from group C) causes theX^(Z)—Z or the X^(D)-D bond to rupture as well. The presence of aself-immolating moiety is desirable in the case of spacer X^(D) because,if, after cleavage of the conjugate, spacer X^(D) or a portion thereofwere to remain attached to D, the biological activity of D may beimpaired. The use of a self-immolating moiety is especially desirablewhere cleavable group C is a polypeptide, in which instance theself-immolating moiety typically is located adjacent thereto, in orderto prevent D from sterically or electronically interfering with peptidecleavage.

Exemplary self-immolating moieties (i)-(v) bonded to a hydroxyl or aminogroup of D are shown below:

The self-immolating moiety is the structure between dotted lines a and b(or dotted lines b and c), with adjacent structural features shown toprovide context. Self-immolating moieties (i) and (v) are bonded to aD-NH₂ (i.e., conjugation is via an amino group), while self-immolatingmoieties (ii), (iii), and (iv) are bonded to a D-OH (i.e., conjugationis via a hydroxyl or carboxyl group). Cleavage of the bond at dottedline b by an enzyme—a peptidase in the instance of structures (i)-(v)and a β-glucuronidase in the instance of structure (vi)—initiates aself-immolating reaction sequence that results in the cleavage of thebond at dotted line a and the consequent release of D-OH or D-NH₂, asthe case may be. By way of illustration, self-immolating mechanisms forstructures (i) and (iv) are shown below:

In other words, cleavage of a first chemical bond at one part of aself-immolating group initiates a sequence of steps that results in thecleavage of a second chemical bond—the one connecting theself-immolating group to the drug—at a different part of theself-immolating group, thereby releasing the drug.

In some instances, self-immolating groups can be used in tandem, asshown by structure (vii). In such case, cleavage at dotted line ctriggers self-immolation of the moiety between dotted lines b and c by a1,6-elimination reaction, followed by self-immolation of the moietybetween dotted lines a and b by a cyclization-elimination reaction. Foradditional disclosures regarding self-immolating moieties, see Carl etal., J. Med. Chem. 1981, 24, 479; Carl et al., WO 81/01145 (1981);Dubowchik et al., Pharmacology & Therapeutics 1999, 83, 67; Firestone etal., U.S. Pat. No. 6,214,345 B1 (2001); Toki et al., J. Org. Chem. 2002,67, 1866; Doronina et al., Nature Biotechnology 2003, 21, 778 (erratum,p. 941); Boyd et al., U.S. Pat. No. 7,691,962 B2; Boyd et al., US2008/0279868 A1; Sufi et al., WO 2008/083312 A2; Feng, U.S. Pat. No.7,375,078 B2; Jeffrey et al., U.S. Pat. No. 8,039,273; and Senter etal., US 2003/0096743 A1; the disclosures of which are incorporated byreference.

In another embodiment, Z and D are linked by a non-cleavable linker,i.e., C is absent. Metabolism of D eventually reduces the linker to asmall appended moiety that does not interfere with the biologicalactivity of D.

Conjugation Techniques

Conjugates of TLR7 agonists disclosed herein preferably are made byfirst preparing a compound comprising D and linker(X^(D))_(a)(C)_(c)(X^(Z))_(b) (where X^(D), C, X^(Z), a, b, and c are asdefined for formula (II)) to form drug-linker compound represented byformula (III):D-(X^(D))_(a)(C)_(c)(X^(Z))_(b)—R³¹  (III)where R³¹ is a functional group suitable for reacting with acomplementary functional group on Z to form the conjugate. Examples ofsuitable groups R³¹ include amino, azide, thiol, cyclooctyne,

where R³² is Cl, Br, F, mesylate, or tosylate and R³³ is Cl, Br, I, F,OH, —O—N-succinimidyl, —O-(4-nitrophenyl), —O-pentafluorophenyl, or—O-tetrafluorophenyl. Chemistry generally usable for the preparation ofsuitable moieties D-(X^(D))_(a)C(X^(Z))_(b)—R³¹ is disclosed in Ng etal., U.S. Pat. No. 7,087,600 B2 (2006); Ng et al., U.S. Pat. No.6,989,452 B2 (2006); Ng et al., U.S. Pat. No. 7,129,261 B2 (2006); Ng etal., WO 02/096910 A1; Boyd et al., U.S. Pat. No. 7,691,962 B2; Chen etal., U.S. Pat. No. 7,517,903 B2 (2009); Gangwar et al., U.S. Pat. No.7,714,016 B2 (2010); Boyd et al., US 2008/0279868 A1; Gangwar et al.,U.S. Pat. No. 7,847,105 B2 (2010); Gangwar et al., U.S. Pat. No.7,968,586 B2 (2011); Sufi et al., U.S. Pat. No. 8,461,117 B2 (2013); andChen et al., U.S. Pat. No. 8,664,407 B2 (2014); the disclosures of whichare incorporated herein by reference.

Preferably reactive functional group —R³¹ is —NH₂, —OH, —CO₂H, —SH,maleimido, cyclooctyne, azido (—N₃), hydroxylamino (—ONH₂) orN-hydroxysuccinimido. Especially preferred functional groups —R³¹ are:

An —OH group can be esterified with a carboxy group on the antibody, forexample, on an aspartic or glutamic acid side chain.

A —CO₂H group can be esterified with a —OH group or amidated with anamino group (for example on a lysine side chain) on the antibody.

An N-hydroxysuccinimide group is functionally an activated carboxylgroup and can conveniently be amidated by reaction with an amino group(e.g., from lysine).

A maleimide group can be conjugated with an —SH group on the antibody(e.g., from cysteine or from the chemical modification of the antibodyto introduce a sulfhydryl functionality), in a Michael additionreaction.

Where an antibody does not have a cysteine —SH available forconjugation, an ε-amino group in the side chain of a lysine residue canbe reacted with 2-iminothiolane orN-succinimidyl-3-(2-pyridyldithio)propionate (“SPDP”) to introduce afree thiol (—SH) group—creating a cysteine surrogate, as it were. Thethiol group can react with a maleimide or other nucleophile acceptorgroup to effect conjugation. The mechanism if illustrated below with2-iminothiolane.

Typically, a thiolation level of two to three thiols per antibody isachieved. For a representative procedure, see Cong et al., U.S. Pat. No.8,980,824 B2 (2015), the disclosure of which is incorporated herein byreference.

In a reversed arrangement, an antibody Z can be modified withN-succinimidyl 4-(maleimidomethyl)-cyclohexanecarboxylate (“SMCC”) orits sulfonated variant sulfo-SMCC, both of which are available fromSigma-Aldrich, to introduce a maleimide group thereto. Then, conjugationcan be effected with a drug-linker compound having an —SH group on thelinker.

An alternative conjugation method employs copper-free “click chemistry,”in which an azide group adds across a strained cyclooctyne to form an1,2,3-triazole ring. See, e.g., Agard et al., J. Amer. Chem. Soc. 2004,126, 15046; Best, Biochemistry 2009, 48, 6571, the disclosures of whichare incorporated herein by reference. The azide can be located on theantibody and the cyclooctyne on the drug-linker moiety, or vice-versa. Apreferred cyclooctyne group is dibenzocyclooctyne (DIBO). Variousreagents having a DIBO group are available from Invitrogen/MolecularProbes, Eugene, Oreg. The reaction below illustrates click chemistryconjugation for the instance in which the DIBO group is attached to theantibody (Ab):

Yet another conjugation technique involves introducing a non-naturalamino acid into an antibody, with the non-natural amino acid providing afunctionality for conjugation with a reactive functional group in thedrug moiety. For instance, the non-natural amino acidp-acetylphenylalanine can be incorporated into an antibody or otherpolypeptide, as taught in Tian et al., WO 2008/030612 A2 (2008). Theketone group in p-acetylphenyalanine can be a conjugation site via theformation of an oxime with a hydroxylamino group on the linker-drugmoiety. Alternatively, the non-natural amino acid p-azidophenylalaninecan be incorporated into an antibody to provide an azide functionalgroup for conjugation via click chemistry, as discussed above.Non-natural amino acids can also be incorporated into an antibody orother polypeptide using cell-free methods, as taught in Goerke et al.,US 2010/0093024 A1 (2010) and Goerke et al., Biotechnol. Bioeng. 2009,102 (2), 400-416. The foregoing disclosures are incorporated herein byreference. Thus, in one embodiment, an antibody that is used for makinga conjugate has one or more amino acids replaced by a non-natural aminoacid, which preferably is p-acetylphenylalanine or p-azidophenylalanine,more preferably p-acetylphenylalanine.

Still another conjugation technique uses the enzyme transglutaminase(preferably bacterial transglutaminase from Streptomyces mobaraensis orBTG), per Jeger et al., Angew. Chem. Int. Ed. 2010, 49, 9995. BTG formsan amide bond between the side chain carboxamide of a glutamine (theamine acceptor) and an alkyleneamino group (the amine donor), which canbe, for example, the ε-amino group of a lysine or a 5-amino-n-pentylgroup. In a typical conjugation reaction, the glutamine residue islocated on the antibody, while the alkyleneamino group is located on thelinker-drug moiety, as shown below:

The positioning of a glutamine residue on a polypeptide chain has alarge effect on its susceptibility to BTG mediated transamidation. Noneof the glutamine residues on an antibody are normally BTG substrates.However, if the antibody is deglycosylated—the glycosylation site beingasparagine 297 (N297; numbering per EU index as set forth in Kabat etal., “Sequences of proteins of immunological interest,” 5th ed., Pub.No. 91-3242, U.S. Dept. Health & Human Services, NIH, Bethesda, Md.,1991; hereinafter “Kabat”) of the heavy chain—nearby glutamine 295(Q295) is rendered BTG susceptible. An antibody can be deglycosylatedenzymatically by treatment with PNGase F (Peptide-N-Glycosidase F).Alternatively, an antibody can be synthesized glycoside free byintroducing an N297A mutation in the constant region, to eliminate theN297 glycosylation site. Further, it has been shown that an N297Qsubstitution not only eliminates glycosylation, but also introduces asecond glutamine residue (at position 297) that too is an amineacceptor. Thus, in one embodiment, the antibody is deglycosylated. Inanother embodiment, the antibody has an N297Q substitution. Thoseskilled in the art will appreciate that deglycosylation bypost-synthesis modification or by introducing an N297A mutationgenerates two BTG-reactive glutamine residues per antibody (one perheavy chain, at position 295), while an antibody with an N297Qsubstitution will have four BTG-reactive glutamine residues (two perheavy chain, at positions 295 and 297).

An antibody can also be rendered susceptible to BTG-mediated conjugationby introducing into it a glutamine containing peptide, or “tag,” astaught, for example, in Pons et al., US 2013/0230543 A1 (2013) andRao-Naik et al., WO 2016/144608 A1.

In a complementary approach, the substrate specificity of BTG can bealtered by varying its amino acid sequence, such that it becomes capableof reacting with glutamine 295 in an umodified antibody, as taught inRao-Naik et al., WO 2017/059158 A1 (2017).

While the most commonly available bacterial transglutaminase is thatfrom S. mobaraensis, transglutaminase from other bacteria, havingsomewhat different substrate specificities, can be considered, such astransglutaminase from Streptoverticillium ladakanum (Hu et al., US2009/0318349 A1 (2009), US 2010/0099610 A1 (2010), and US 2010/0087371A1 (2010)).

TLR7 agonists of this disclosure having a primary or secondary alkylamine are particularly suitable for use in conjugates, as the secondaryamine provides a functional group for attachment of the linker. Anexample of such a TLR7 agonist-linker compound is compound 19, whichcontains an enzymatically cleavable linker. FIG. 4 shows a schemeaccording to which compound 19 can be prepared.

An example of a TLR7 agonist-linker compound that contains anon-enzymatically cleavable linker is compound 21. FIG. 5 shows a schemefor synthesizing compound 21.

Both compounds 19 and 21 contain a primary alkylamino group, renderingthem amenable to conjugation with transglutaminase. A suitableconjugation procedure is described in the Examples hereinbelow.

Conjugation can also be effected using the enzyme Sortase A, as taughtin Levary et al., PLoS One 2011, 6(4), e18342; Proft, Biotechnol. Lett.2010, 32, 1-10; Ploegh et al., WO 2010/087994 A2 (2010); and Mao et al.,WO 2005/051976 A2 (2005). The Sortase A recognition motif (typicallyLPXTG, where X is any natural amino acid) may be located on the ligand Zand the nucleophilic acceptor motif (typically GGG) may be the group R³¹in formula (III), or vice-versa.

TLR7 Agonist Conjugates

Applying the fore-described techniques, TLR7 agonist conjugates such asthe ones shown below can be prepared:

where m is 1, 2, 3, or 4 and Ab is an antibody.Pegylation

Attachment of a poly(ethylene glycol) (PEG) chain to a drug(“PEGylation”) can improve the latter's pharmacokinetic properties. Thecirculation half-life of the drug is increased, sometimes by over anorder of magnitude, concomitantly reducing the dosage needed to achievea desired therapeutic effect. PEGylation can also decrease metabolicdegradation of a drug and reduce its immunogenicity. For a review, seeKolate et al., J. Controlled Release 2014, 192, 167.

Initially, PEGylation was applied to biologic drugs. As of 2016, overten PEGylated biologics had been approved. Turecek et al., J.Pharmaceutical Sci. 2016, 105, 460. More recently, stimulated by thesuccessful application of the concept to biologics, attention has turnedtowards its application to small molecule drugs. In addition to theaforementioned benefits, PEGylated small molecule drugs may haveincreased solubility and cause fewer toxic effects. Li et al. Prog.Polymer Sci. 2013, 38, 421.

The compounds disclosed herein can be PEGylated. Where a compound has analiphatic hydroxyl or aliphatic primary or secondary amine, such as thecase of compound 6 (FIG. 1) or Ia-05 (arrows), it can be PEGylated viaan ester, amide, carbonate, or carbamate group with a carboxy-containingPEG molecule utilizing conventional techniques such asdicyclohexylcarbodiimide, HATU, N-hydroxysuccinimide esters, and thelike. Various other methods for PEGylating pharmaceutical molecules aredisclosed in Alconcel et al., Polymer Chem. 2011, 2, 1442, thedisclosure of which is incorporated herein by reference.

If desired, a TLR7 agonist disclosed herein can be PEGylated via anenzymatically cleavable linker comprising a self-immolating moiety, toallow release of the un-PEGylated agonist in a designed manner. Further,PEGylation can be combined with conjugation to a protein such as anantibody, if the PEG-containing molecule has a suitable functional groupsuch as an amine for attachment to the protein. The protein can providean additional therapeutic function or, if an antibody, can provide atargeting function. These concepts are illustrated in the followingreaction sequence, where TLR7-NH—R generically represents a TLR7agonist:

In the above reaction sequence, the valine-citrulline (Val-Cit)dipeptide is cleavable by the enzyme cathepsin B, with a p-aminobenzyloxycarbonyl (PABC) group serving as a self-immolating spacer. Thefunctional group for conjugation is an amine group, which is temporarilyprotected by an Fmoc group. Conjugation is effected by the enzymetransglutaminase, with a glutamine (Gin) side chain acting as the acylacceptor. The subscript x, denoting the number of PEG repeat units, canvary widely, depending on the purpose of the PEGylation, as discussedbelow. For some purposes, x can be relatively small, such as 2, 4, 8,12, or 24. For other purposes, x is large, for example between about 45and about 910.

Those skilled in the art will understand that the sequence isillustrative and that other elements—peptide, self-immolating group,conjugation method, PEG length, etc.—may be employed, as is well knownin the art. They will also understand that, while the above sequencecombines PEGylation and conjugation, PEGylation does not requireconjugation, and vice-versa.

Where the compound lacks aliphatic hydroxyl or aliphatic primary orsecondary amine, as in the case of compound 7 (FIG. 1), it still can bePEGylated at the aromatic amine (arrow). A method for PEGylating at thisposition is disclosed by Zarraga, US 2017/0166384 A1 (2007), thedisclosure of which is incorporated by reference.

In some embodiments, it may be desirable to have multiple PEGylatedagonists linked in a single molecule. For instance, four PEGylated armscan be constructed on pentaerythritol (C(CH₂OH)₄) and a TLR7 agonist canbe attached to each PEGylated arm. See Gao et al., US 2013/0028857 A1(2013), the disclosure of which is incorporated by reference.

For modulating pharmacokinetics, it is generally preferred that the PEGmoiety have a formula weight of between about 2 kDa (corresponding toabout 45 —(CH₂CH₂O)— repeating units) and between about 40 kDa(corresponding to about 910 —(CH₂CH₂O)— repeating units), morepreferably between about 5 kDa and about 20 kDa. That is, the range ofthe subscript x in the above formulae is from about 45 to about 910. Itis to be understood that PEG compositions are not 100% homogeneous but,rather, exhibit a distribution of molecular weights. Thus, a referenceto, for example, “20 kDa PEG” means PEG having an average molecularweight of 20 kDa.

PEGylation can also be used for improving the solubility of an agonist.In such instances a shorter PEG chain can be used, for examplecomprising 2, 4, 8, 12, or 24 repeating units.

EXAMPLES

The practice of this invention can be further understood by reference tothe following examples, which are provided by way of illustration andnot of limitation.

Example 1—Synthesis of Formula (Ia) Compounds

This example and FIG. 1 relate to the synthesis of compounds accordingto formula (Ia).

A suspension of pyrazine-2,5-dicarboxylic acid 1 (5 g, 29.7 mmol) andHCl (1.25 M in MeOH, 50 mL, 62.5 mmol) was stirred at 60° C. for 20 h,after which the reaction was complete. The reaction mixture wasconcentrated on a rotary evaporator. The crude product was suspended insaturated NaHCO₃, extracted with 10% MeOH in dichloromethane (DCM, 3×150mL). The combined organic extracts were dried with Na₂SO₄, filtered andconcentrated to yield dimethyl pyrazine-2,5-dicarboxylate (4.37 g, 22.28mmol, 74.9% yield). LCMS ESI: calculated for C₈H₈N₂O₄=197.0 (M+H⁺),found 197.0 (M+H⁺).

A stirred suspension of dimethyl pyrazine-2,5-dicarboxylate (4.32 g,22.02 mmol) in MeOH (103 mL) and DCM (44.0 mL) was treated with NaBH₄(0.833 g, 22.02 mmol) at 0° C. After 1 h, LCMS indicated 70% conversionwith major mono-alcohol product and minor bis-alcohol product. MoreNaBH₄ (100 mg, 4.34 mmol) was added at 0° C. and stirring continued foranother 45 min. No more starting material was detected by LCMS. Thereaction mixture was quenched by slowly adding half-saturated NH₄Cl andextracted with EtOAc (3×100 mL). The combined organic extracts weredried over Na₂SO₄, filtered and concentrated. The crude product waspurified on a 24 g silica column, eluted with 20% MeOH in DCM. Thedesired fractions were concentrated to yield methyl5-(hydroxymethyl)pyrazine-2-carboxylate (2.43 g, 14.45 mmol, 65.6%yield). LCMS ESI: calculated for C₇H₈N₂O₃=169.1 (M+H⁺), found 169.0(M+H⁺).

A solution of methyl 5-(hydroxymethyl)pyrazine-2-carboxylate (1.65 g,9.81 mmol) in tetrahydrofuran (THF, 49.1 mL) was treated withtriphenylphosphine (3.09 g, 11.78 mmol), followed by N-bromosuccinimide(NBS, 2.096 g, 11.78 mmol) at room temperature (RT). After stirring for90 min, the reaction was complete. After quenching with water andextraction with EtOAc (3×50 mL), the combined organic extracts weredried over Na₂SO₄, filtered and concentrated. The crude product waspurified on a 40 silica column, eluted with EtOAc:Hexane (0-100%gradient). The desired fractions were concentrated and yield compound 2(1.28 g, 5.54 mmol, 56.5% yield). LCMS ESI: calculated forC₇H₇BrN₂O₂=230.0, 232.0 (M+H⁺), found 230.9, 232.9 (M+H⁺).

To a suspension of compound 3 in trifluoroacetic acid (TFA, CAS Reg. No.866268-31-7, prepared according to WO 2011/049815 A1, 1.824 g, 5.19mmol)nd cesium carbonate (5.42 g, 16.62 mmol) in DMF (20 mL) was addedcompound 2 (1.2 g, 5.19 mmol). The reaction mixture was stirred at RTfor 1 h, after which the reaction was complete. After quenching withwater, the resulting solid was collected by filtration and rinsed withwater and air dried in vacuo to yield compound 4 (1.67 g, 4.31 mmol, 83%yield) which was carried over to next step without further purification.LCMS ESI: calculated for C₁₇H₂₁H₇O₄=388.2 (M+H⁺), found 388.1 (M+H⁺). ¹HNMR (400 MHz, CHLOROFORM-d) δ 9.20 (d, J=1.3 Hz, 1H), 8.68 (s, 1H), 5.36(s, 2H), 4.33-4.27 (m, 2H), 4.11 (s, 3H), 4.04 (s, 2H), 1.77-1.70 (m,2H), 1.46 (br d, J=7.7 Hz, 2H), 0.93 (t, J=7.5 Hz, 3H).

A stirred solution of compound 4 (1.06 g, 2.64 mmol) in THF (10 mL) wastreated dropwise with lithium aluminum hydride (1.0 M in THF, 3.96 mL,3.96 mmol) at 0° C. After stirring for 3 h, LCMS indicated reaction wascomplete. Na₂SO₄.10H₂O was added and the reaction mixture was stirredfor 1 h at RT. The solid was filtered off and rinsed with MeOH and thefiltrate was concentrated. The crude product was purified on a 40 gsilica column, eluted with 20% MeOH in DCM (0-40% gradient). The desiredfractions were concentrated to yield compound 5 (487 mg, 1.355 mmol,51.3% yield). LCMS ESI: calculated for C₁₆H₂₁H₇O₃=359.2 (M+H⁺), found360.1 (M+H⁺). ¹H NMR (400 MHz, DMSO-d₆) δ 9.91 (s, 1H), 8.48 (d, J=5.5Hz, 2H), 6.68-6.00 (m, 2H), 4.96 (s, 2H), 4.52 (s, 2H), 4.01 (t, J=6.6Hz, 2H), 1.58-1.42 (m, 2H), 1.32-1.19 (m, 2H), 0.80 (t, J=7.4 Hz, 3H).

Compound 5 (12.7 mg, 0.035 mmol) was dissolved in 0.5 mL THF and 1.0 MHCl (1.0 mL). After stirring at 60° C. for 3 h, the reaction wascomplete. The reaction mixture was concentrated. The crude product waspurified on a 15.5 g C18 Aq column, eluting with 0.05% TFA inacetonitrile:0.05% TFA in H₂O (0-50% gradient) to yield compound 6 (4.0mg, 10.89 μmol, 10.54% yield). LCMS ESI: calculated for C₁₅H₁₉N₇O₃=346.2(M+H⁺), found 346.1 (M+H⁺). ¹H NMR (400 MHz, DMSO-d₆) δ 9.91 (s, 1H),8.48 (d, J=5.5 Hz, 2H), 6.68-6.00 (m, 2H), 4.96 (s, 2H), 4.52 (s, 2H),4.01 (t, J=6.6 Hz, 2H), 1.58-1.42 (m, 2H), 1.32-1.19 (m, 2H), 0.80 (t,J=7.4 Hz, 3H).

A suspension of compound 6 (120 mg, 0.347 mmol) in THF (2 mL) wastreated with thionyl chloride (0.254 ml, 3.47 mmol) at RT. Afterstirring for 45 min, the reaction was complete. The thionyl chloride wasazeotropically removed with DCM (three times). The crude chloromethylcompound 7 was carried over to next step as-is. LCMS ESI: calculated forC₁₅H₁₈ClN₇O₂=363.1 (M+H⁺), found 364.0 (M+H⁺).

A solution of compound 7 (10 mg, 0.027 mmol) in N,N-Dimethylformamide(DMF, 1 mL) was treated with cyclobutanamine (0.012 mL, 0.137 mmol) andthen stirred at 60° C. for 1 h. LCMS indicated the reaction wascomplete. The reaction mixture was then purified on a 15.5 g C18 Aqcolumn, eluting with 0.05% TFA in acetonitrile:0.05% TFA in H₂O (0-50%gradient) to yield compound Ia-08 (3.7 mg, 9.10 μma 33.1% yield). LCMSESI: calculated for C₁₉H₂₆H₈O₂=397.2 (M−H⁺), found 397.2 (M−H⁺). ¹H NMR(400 MHz, METHANOL-d₄) δ 8.65 (s, 1H), 8.56 (s, 1H), 5.21 (s, 2H), 4.22(t, J=6.5 Hz, 2H), 3.99 (s, 2H), 3.54-3.42 (m, 2H), 2.28-2.18 (m, 2H),2.00-1.89 (m, 2H), 1.84-1.65 (m, 4H), 1.53-1.41 (m, 3H), 0.97 (t, J=7.4Hz, 4H).

By generally following the above procedure and by using alternativeamines to cyclobutanamine, additional compounds according to formula(Ia) were prepared, as listed in Table D below.

TABLE D Additional compounds (Ia) Mass Spectrum Compound Expected massObserved mass Number Amine (M + H) (M + H) Ia-01

436.2 436.1 Ia-02

415.2 415.1 Ia-03

435.2 435.1 Ia-04

403.2 403.2 Ia-05

383.2 (M − H) 383.0 (M − H) Ia-06

385.2 (M − H) 385.2 (M − H) Ia-07

399.2 (M − H) 399.2 (M − H) Ia-09

399.2 (M − H) 399.1 (M − H) Ia-10

359.2 359.1 Ia-11

388.1 (M − H) 387.2 (M − H) Ia-12

646.4 646.4

Even though it was used in the scheme FIG. 1 as a syntheticintermediate, compound 6 also possesses TLR7 agonist activity, with anEC₅₀ of 288 nM.

Example 2—Synthesis of Formula (Ic) Compounds

This example and FIG. 2 relate to the synthesis of compounds accordingto formula (Ic).

To a solution of dimethyl pyridine-2,5-dicarboxylate 8 (CAS Reg. No.881-86-7, 5 g, 25.6 mmol) in THF (50 mL)/MeOH (100 mL) was added calciumchloride (11.37 g, 102 mmol). The reaction mixture was sonicated for 5min and cooled to 0° C. Sodium borohydride (2.423 g, 64.0 mmol) powderwas added slowly (gas evolution). The mixture was stirred at 0° C. for 2h, after which LCMS showed completion of the reaction. The reaction wasquenched by addition of ice and extracted with CHCl₃ and dried overNa₂SO₄ to provide compound 9 as an off-white solid (93% yield). LCMSESI: calculated for C₈H₉NO₃=167.05 (M+H⁺), found 168.0 (M+H⁺).

A solution of compound 9 (2.5 g, 14.96 mmol) in DMF (10 mL) was treatedwith imidazole (1.527 g, 22.43 mmol) and t-butyldimethylsilyl chloride(TBS-Cl, 2.480 g, 16.45 mmol). After 2 h, LCMS showed completion ofreaction. The reaction was washed with sat. aq. NaHCO₃ and brine, andthe organic layer was dried over Na₂SO₄. The crude methyl6-(((tert-butyldimethylsilyl)oxy)methyl)nicotinate (93% yield) was takento next step without further purification.

A solution of the crude methyl6-(((tert-butyldimethylsilyl)oxy)methyl)nicotinate (3.9 g, 13.86 mmol)in THF (50 mL) was cooled to 0° C. and treated dropwise with a solutionof Red-Al™ (sodium bis(2-methoxyethoxy)aluminum hydride solution, 10.37mL, 31.9 mmol). The reaction was stirred for 30 min, after which LCMSshowed completion of the reaction. The reaction was quenched by dropwiseaddition of MeOH (10 mL) followed by 1 M solution of NaOH (20 mL). Themixture was stirred for 20 min. The layers were separated and theaqueous layer was extracted with EtOAc 3 times. Concentration of organicphases provided the desired(6-(((tert-butyldimethylsily)oxy)methyl)pyridin-3-yl)methanol as ayellowish liquid (97% yield).

A mixture of the preceding product (3.4 g, 13.42 mmol) andtriphenylphosphine (3.87 g, 14.76 mmol) in DCM (50 mL) was slowlytreated with N-bromosuccinimide (NBS, 2.63 g, 14.76 mmol) and stirredfor 30 min. LCMS showed the completion of reaction. The solvent wasevaporated and the crude product was purified on 80 g gold silica geleluting with 0-50% EtOAc/hexane to provide compound 10 as white solid(75% yield). LCMS ESI calculated for C₁₃H₂₂BrNOSi=315.06 (M+H⁺), found318.0 (M+H⁺).

A mixture of compound 11 (CAS Reg. No. 473930-51-7, 1.906 g, 9.20 mmol),compound 10 (3.2 g, 10.12 mmol) and cesium carbonate (3.30 g, 10.12mmol) in DMF (20 mL) was heated at 70° C. for 5 h, after which LCMSshowed the completion of reaction. The reaction was filtered to removethe cesium carbonate and the filtrate was diluted with EtOAc (50 mL).The organic layer was washed with water (2×50 mL) and brine (2×50 mL).The solvent was evaporated and the crude product was purified on 80 ggold silica gel column eluting with 0-50% MeOH/DCM to yield compound 12as white solid (56% yield). ¹H NMR (400 MHz, Chloroform-d) δ 8.45 (d,J=2.2 Hz, 1H), 7.55 (dd, J=8.1, 2.3 Hz, 1H), 7.51 (s, 1H), 7.39 (d,J=8.0 Hz, 1H), 5.50 (s, 1H), 5.17 (s, 2H), 4.71 (s, 2H), 4.24 (t, J=6.6Hz, 2H), 1.74-1.63 (m, 2H), 1.47-1.33 (m, 2H), 0.85 (d, J=12.3 Hz, 12H),0.00 (s, 6H). LCMS ESI: calculated for C₂₂H₃₄N₆O₂Si=442.2 (M+H⁺), found443.2 (M+H⁺).

A solution of compound 12 (2.0 g, 4.52 mmol) and sodium acetate (1.853g, 22.59 mmol) in CHCl₃ (20 mL)/THF (10 mL) at 0° C. was treateddropwise with bromine (0.466 mL, 9.04 mmol). After 30 min, LCMS showedcompletion of reaction. The reaction was quenched with 10% aq. sodiumthiosulfate solution and extracted with DCM. The solvent was evaporatedto give the desired brominated intermediate as white solid.

A solution of the preceding brominated intermediate (1.7 g, 3.26 mmol)in MeOH (20 mL) was treated with lithium methanolate (1.238 g, 32.6mmol) and heated at 60° C. overnight. LCMS showed the displacement ofbromide and removal of the TBS group. The solvent was evaporated and thecrude product was taken to next step without further purification.

A solution of the product of the preceding reaction (1.168 g, 3.26 mmol)in MeOH (5 mL) was treated with HCl in water (8.15 mL, 48.9 mmol). Thereaction mixture was heated at 60° C. overnight. LCMS showed thecompletion of the reaction. The solvent was evaporated and the residuewas treated with HCl in water (8.15 mL, 48.9 mmol). The mixture washeated at 60° C. for 2 h and was neutralized with 10 M aqeous NaOH. Theproduct precipitated out and was washed with water and dried under highvacuum to give compound 13 as white solid (47% yield over 3 steps). ¹HNMR (400 MHz, DMSO-d6) δ 10.04 (s, 1H), 8.39 (d, J=2.2 Hz, 2H), 7.62(dd, J=8.0, 2.3 Hz, 2H), 7.35 (d, J=8.0 Hz, 2H), 6.42 (s, 3H), 5.30 (s,2H), 4.80 (s, 4H), 4.45 (d, J=4.3 Hz, 4H), 4.07 (t, J=6.6 Hz, 4H),1.62-1.49 (m, 4H), 1.38-1.24 (m, 4H), 0.84 (t, J=7.4 Hz, 6H). LCMS ESI:calculated for C₁₆H₂₀N₆O₃=344.06 (M+H⁺), found 34.1 (M+H⁺).

A solution of compound 13 (240 mg, 0.697 mmol) in THF (1 mL) was treatedwith thionyl chloride (0.127 mL, 1.742 mmol). The suspension wassonicated for 30 min. LCMS showed the completion of reaction. Thesolvent was evaporated and the crude product 14 (quantitative yield) wastaken to next step.

A solution of crude product 14 (10 mg, 0.028 mmol) in DMF (0.5 mL) wastreated with cyclobutanamine (9.80 mg, 0.138 mmol) in a sealed tube,which was then was heated at 70° C. for 30 min. LCMS showed completionof the reaction. The crude product was directly injected into a Shimadzuprep HPLC with xBridge PrepC18 5 19×150 mm column and eluted with 0-95%MeCN/H2O (0.1% FA) and the product containing fractions were lyophilizedto provide compound Ic-04 (30% yield, 4.5 mg, 10.76 μmol, 39.0% yield)as white solid. ¹H NMR (400 MHz, DMSO-d6) δ 8.38 (d, J=2.2 Hz, 1H), 7.57(dd, J=8.0, 2.3 Hz, 1H), 7.29 (d, J=8.0 Hz, 1H), 6.50 (s, 2H), 4.79 (s,2H), 4.07 (t, J=6.6 Hz, 2H), 3.58 (s, 2H), 3.06 (p, J=7.5 Hz, 1H), 1.96(ddt, J=10.6, 8.3, 4.4 Hz, 2H), 1.65-1.37 (m, 6H), 1.37-1.24 (m, 2H),0.84 (t, J=7.4 Hz, 3H). LCMS ESI: calculated for C₂₀H₂₇N₇O₂=397.2(M+H⁺), found 398.1 (M+H⁺).

By generally following the above procedure and by using alternativeamines to cyclobutanamine, additional compounds according to formula(Ic) were prepared, as listed in Table E below.

TABLE E Additional Compounds (Ic) Mass Spectrum Compound Expected. massObserved mass Number Amine (M + H) (M + H) Ic-01

400.2 400.1 Ic-02

402.2 402.1 Ic-03

384.2 384.1 Ic-04

398.2 398.1 Ic-05

401.2 401.1 Ic-06

434.2 434.1 Ic-07

449.5 449.2 Ic-08

647.7 647.3 Ic-09

428.2 428.2 Ic-10

386.2 386.2 Ic-11

412.2 412.2 Ic-12

427.2 427.2 Ic-13

441.2 441.2

Example 3—Synthesis of Formula (Ib) Compounds

Compounds according to formula (Ib) were prepared analogously to themethods for preparing compounds (Ic), but using as an intermediatechloride 17. The synthesis of chloride 17 is shown schematically in FIG.3, starting from commercially available compound 15 (CAS Reg. No.49668-90-8), proceeding analogously to the preceding example but mutatismutandis.

Analytical data for compounds according to formula (Ib) are provided inTable F below.

TABLE F Compounds (Ib) Mass Spectrum Compound Expected. mass Observedmass Number Amine (M + H) (M + H) Ib-01

400.2 400.2 Ib-02

402.2 402.1 Ib-03

384.2 384.1 Ib-04

398.2 398.1 Ib-05

401.2 401.1 Ib-06

434.2 434.1 Ib-07

449.2 449.2 Ib-08

647.3 647.3

Example 4—Assay for TLR7 Agonist Activity

This example describes a method for assaying TLR7 agonist activity ofthe compounds disclosed in this specification.

Engineered human embryonic kidney blue cells (HEK-Blue™ TLR cells;Invivogen) possessing a human TLR7-secreted embryonic alkalinephosphatase (SEAP) reporter transgene were suspended in a non-selective,culture medium (DMEM high-glucose (Invitrogen), supplemented with 10%fetal bovine serum (Sigma)). HEK-Blue™ TLR7 cells were added to eachwell of a 384-well tissue-culture plate (15,000 cells per well) andincubated 16-18 h at 37° C., 5% CO₂. Compounds (100 nl) were dispensedinto wells containing the HEK-Blue™ TLR cells and the treated cells wereincubated at 37° C., 5% CO₂. After 18 h treatment ten microliters offreshly-prepared Quanti-Blue™ reagent (Invivogen) was added to eachwell, incubated for 30 min (37° C., 5% CO₂) and SEAP levels measuredusing an Envision plate reader (OD=620 nm). The half maximal effectiveconcentration values (EC₅₀; compound concentration which induced aresponse halfway between the assay baseline and maximum) werecalculated.

A representative EC₅₀ assay curve, for compound (Ia-09), is shown inFIG. 6.

Example 5—IL-6 Induction

This example describes a method for assaying interleukin 6 induction bycompounds disclosed in this specification.

Compounds diluted in DMSO were transferred to individual wells of aMatrix Technologies clear, V-bottom 384-well plate using ECHO acousticliquid handling technology (25 nL per well). Human whole-blood samples(25 uL) were added to each well using a CyBio FeliX liquid handlinginstrument. The plate was shaken on a plate shaker for three min beforeincubating the reaction mixtures at 37° C. for 20 h. Basel RPMI 1640medium (supplemented with L-glutamine) was then added to each well (25uL per well) prior to liberating plasma from each sample bycentrifugation (450×g, 5 min, ambient temperature). Treated plasmasamples (3 uL) were subsequently transferred to individual wells of awhite, shallow, 384-well ProxiPlate (Perkin Elmer) using the FeliXliquid handling instrument and their interleukin 6 levels were measuredusing AlphaLISA technology as described by the manufacturer,PerkinElmer. Data analyses software was used to determine compound EC₅₀values where the baseline was established using average DMSO values and100% induction established using reference compound values at thehighest concentration tested. EC₅₀'s can be determined with softwaresuch as Graphpad Prism™.

Example 6—Transglutaminase Mediated Conjugation

The following procedure can be used for transglutaminase mediatedconjugation of agonist-linker compounds wherein the linker has an aminegroup that can act as an amine donor. The antibody can be one that has atransglutaminase-reactive glutamine, for example one with an N297A orN297Q substitution. Conjugation is carried out by recombinant bacterialtransglutaminase with a molar ratio of antibody:enzyme of 5:1. Theconjugation is carried out using standard protocols in 50 mM Trisbuffer, pH 8.0, incubated overnight at 37° C. The resulting conjugate ispurified on a Protein A column, pre-equilibrated with 50 mM Tris, pH8.0. The conjugate is eluted with 0.1 M sodium citrate buffer, pH 3.5.The eluted fractions are neutralized with 1M Tris pH 9.0. The conjugatecan be formulated in 20 mg/mL Sorbitol, 10 mg/mL Glycine, pH 5.0.

The foregoing detailed description of the invention includes passagesthat are chiefly or exclusively concerned with particular parts oraspects of the invention. It is to be understood that this is forclarity and convenience, that a particular feature may be relevant inmore than just the passage in which it is disclosed, and that thedisclosure herein includes all the appropriate combinations ofinformation found in the different passages. Similarly, although thevarious figures and descriptions herein relate to specific embodimentsof the invention, it is to be understood that where a specific featureis disclosed in the context of a particular figure or embodiment, suchfeature can also be used, to the extent appropriate, in the context ofanother figure or embodiment, in combination with another feature, or inthe invention in general.

Further, while the present invention has been particularly described interms of certain preferred embodiments, the invention is not limited tosuch preferred embodiments. Rather, the scope of the invention isdefined by the appended claims.

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Full citations for the following references cited in abbreviated fashionby first author (or inventor) and date earlier in this specification areprovided below. Each of these references is incorporated herein byreference for all purposes.

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What is claimed is:
 1. A compound having a structure according toformula (Ia)

wherein R¹ is (C₁-C₅ alkyl)O, (C₁-C₂ alkyl)O(CH₂)₂₋₃O, (C₁-C₅alkyl)C(═O)O, (C₁-C₅ alkyl)NH, (C₁-C₂ alkyl)O(CH₂)₂₋₃NH, or (C₁-C₅alkyl)C(═O)NH; and R³ and R⁴ are independently H; C₁-C₆ alkyl;(CH₂)₂₋₄OH; (CH₂)₂₋₄O(C₁-C₃ alkyl); (CH₂)₂₋₄NH₂; (CH₂)₂₋₄NH(C₁-C₃alkyl); (CH₂)₂₋₄N(C₁-C₃ alkyl)₂; (CH₂)₁₋₃(aryl); (CH₂)₁₋₃(heteroaryl);(CH₂)₂₋₄(OCH₂CH₂)₂₋₈(CH₂)₂₋₄NHBoc;

 wherein a CH₂ group in the cycloaliphatic ring may be replaced by O, S,NH, or N(C₁-C₃ alkyl) and the cycloaliphatic ring may be substitutedwith C₁-C₃ alkyl, OH, O(C₁-C₃ alkyl), halo, (CH₂)₀₋₃NH₂, or(CH₂)₀₋₃NH(C₁-C₃ alkyl);  or R³ and R⁴ combine with the nitrogen towhich they are bonded to form a cyclic amine of the structure

 wherein a CH₂ group in the cycloaliphatic ring that is separated fromthe amine nitrogen by at least two CH₂ groups may be replaced by O, S,NH, or N(C₁-C₃ alkyl) and the cyclic amine may be substituted with C₁-C₃alkyl, OH, O(C₁-C₃ alkyl), halo, (CH₂)₀₋₃NH₂, or (CH₂)₀₋₃NH(C₁-C₃alkyl).
 2. A compound according to claim 1, wherein —N(R³)(R⁴) is


3. A compound according to claim 1, wherein, in formula (Ia), R¹ isn-BuO.
 4. A compound according to claim 1, wherein R⁴ is H and R³ isother than H.
 5. A compound according to claim 1, wherein R¹ is n-BuO orMeOCH₂CH₂O.
 6. A compound according to claim 1, selected from the groupconsisting of compounds (Ia-01) through (Ia-11):